Implantable devices and techniques for the treatment of obesity

ABSTRACT

In an embodiment, the present disclosure pertains to an organ-specific wireless optogenetic device. In some embodiments, the device includes an electronic circuit, such that the electronic circuit is configured to harvest and convert radio frequency (RF) energy into optical energy and a tether having a μLED, where the μLED illuminates targeted regions in the organ. In an additional embodiment, the present disclosure pertains to a method of treating obesity. In general, the method includes implanting an organ-specific wireless optogenetic device into a subject, activating an RF-power system to produce RF energy, harvesting, by the organ-specific wireless optogenetic device, the RF energy, converting, by the organ-specific wireless optogenetic device, the RF energy into optical energy, illuminating, by the μLED, targeted regions in the stomach of the subject, and stimulating nerve endings to thereby suppress appetite in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to, and incorporates byreference the entire disclosure of, U.S. Provisional Patent ApplicationNo. 63/153,937 filed on Feb. 25, 2021.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under EEC-1648451awarded by the Precise Advanced Technologies and Health Systems forUnderserved Populations (PATHS-UP)/National Science Foundation. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates generally to implantable devices and moreparticularly, but not by way of limitation, to implantable devices andtechniques for the treatment of obesity.

BACKGROUND

This section provides background information to facilitate a betterunderstanding of the various aspects of the disclosure. It should beunderstood that the statements in this section of this document are tobe read in this light, and not as admissions of prior art.

The vagus nerve supports diverse autonomic functions and behaviorsimportant for health and survival. To understand how specific componentsof the vagus nerve contribute to behaviors and long-term physiologicaleffects, it is important to modulate their activity with anatomicalspecificity in awake, freely behaving conditions using reliable methods.Here, an organ-specific scalable, multimodal, wireless optoelectronicdevice for precise and chronic optogenetic manipulations in vivo isintroduced. When combined with an advanced, coil-antenna system and amultiplexing strategy for powering 8 individual homecages using a singleRF transmitter, the proposed wireless telemetry enables low cost,high-throughput, and precise functional mapping of peripheral neuralcircuits, including long-term behavioral and physiological measurements.Deployment of these technologies reveals an unexpected role for stomach,non-stretch vagal sensory fibers in suppressing appetite anddemonstrates the durability of the miniature wireless device insideharsh gastric conditions.

Gastric bypass surgery is sometimes the last resort for those whostruggle with obesity or have serious health-related issues due to theirweight. Since this procedure involves making a small stomach pouch andrerouting the digestive tract, it is very invasive and prolongs therecovery period for patients. The devices as disclosed herein can helpwith weight loss, and requires a simpler operative procedure forimplantation. The centimeter-sized device provides a feeling of fullnessby stimulating endings of the vagus nerve with light. Unlike otherdevices that require a power cord, the devices as disclosed herein arewireless and can be controlled externally from a remote radio frequencysource.

SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts that arefurther described below in the Detailed Description. This summary is notintended to identify key or essential features of the claimed subjectmatter, nor is it to be used as an aid in limiting the scope of theclaimed subject matter.

In an embodiment, the present disclosure pertains to an organ-specificwireless optogenetic device. In some embodiments, the device includes anelectronic circuit, such that the electronic circuit is configured toharvest and convert radio frequency (RF) energy into optical energy anda tether having a μLED, where the μLED illuminates targeted regions inthe organ.

In some embodiments, the RF energy is harvested from a remotely locatedRF-power system. In some embodiments, the μLED is positioned at a middlepoint of the tether (e.g., between distal and proximal ends of thetether). In some embodiments, the organ-specific wireless optogeneticdevice is in a pre-curved, sandwiched configuration. In someembodiments, the sandwich configuration has flexible copper/polyimide(Cu/PI) bilayer films. In some embodiments, the sandwiched configurationis PI/Cu/Cu/PI. In some embodiments, a portion of the tether isencapsulated by a composition. In some embodiments, the portion of thetether includes the μLED. In some embodiments, the composition includessilicone. In some embodiments, the composition is coated on the portionof the tether while the tether is in a bent position.

In an additional embodiment, the present disclosure pertains to a methodof treating obesity. In general, the method includes implanting anorgan-specific wireless optogenetic device into a subject, activating anRF-power system to produce RF energy, harvesting, by the organ-specificwireless optogenetic device, the RF energy, converting, by theorgan-specific wireless optogenetic device, the RF energy into opticalenergy, illuminating, by the μLED, targeted regions in the stomach ofthe subject, and stimulating nerve endings to thereby suppress appetitein the subject.

In some embodiments, the organ-specific wireless optogenetic deviceincludes an electronic circuit configured to harvest and convert radiofrequency (RF) energy into optical energy and a tether having a μLEDpositioned at a middle point of the tether.

In some embodiments, the nerve endings are vagus nerve endings. In someembodiments, the RF-power system is remotely-located and has adual-coil-antenna system. In some embodiments, the organ-specificwireless optogenetic device is in a pre-curved, sandwichedconfiguration. In some embodiments, the sandwich configuration includesflexible copper/polyimide (Cu/PI) bilayer films. In some embodiments,the sandwiched configuration is PI/Cu/Cu/PI. In some embodiments, aportion of the tether including the μLED is encapsulated by acomposition. In some embodiments, the composition includes silicone. Insome embodiments, the composition is coated on the portion of the tetherwhile the tether is in a bent position. In some embodiments, appetitesuppression occurs via a negative valence mechanism that alters tastepreferences.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the subject matter of the presentdisclosure may be obtained by reference to the following DetailedDescription when taken in conjunction with the accompanying Drawingswherein:

FIG. 1 is an illustration of a soft, wireless gastric optogeneticimplant with a pre-curved, sandwiched tether, according to embodimentsof the disclosure;

FIGS. 2a-2e illustrate a procedure for fabricating a soft, wirelessgastric optogenetic implant with a pre-curved, sandwiched tether,according to embodiments of the disclosure;

FIGS. 3a-3d are plots illustrating various performance parameters of asoft, wireless gastric optogenetic implant with a pre-curved, sandwichedtether. FIG. 3a is a plot of output power vs. curvatures of a tether.The legend numbers represent the curvature of the device as the lengthof the arc for a radius of 2 mm, smaller numbers represent sharperU-shape angle. FIGS. 3b and 3c show measurement results of devicelifetime cycling test for both structures when strain applied in thehorizontal (FIG. 3b ) and vertical direction (FIG. 3c ). FIG. 3d showsmeasurements of device lifetime for the pre- and post-curved structurewhen implanted (pre-curved, n=8; post-curved, n=8). Bar graphs aremean±SEM. Statistical comparison was made using two-tailed t test;***p<0.001.

FIGS. 4a and 4b illustrate optogenetic targeting of Calca+vagalafferents in the stomach. FIG. 4a shows light intensity measurementscomparing LED implantation inside vs. outside the stomach (n=5, p<0.01),with varying RF powers (p<0.001). Dashed horizontal lines indicate lightintensity needed for 10 and 50% maximal activation of channelrhodopsin2.FIG. 4b shows comparison of total food intake, number of meals, and mealsize in mice implanted with LED device (n=7) or sham operated (n=6)(p=0.71). Bar graphs are mean±SEM. Statistical comparisons were madeusing two-way repeated-measures ANOVA, Tukey's post hoc; ***p<0.001.

FIG. 5a-5d illustrates activation of Calca+stomach vagal afferentssuppress appetite via negative valence mechanism. FIG. 5a showsfrequency-dependent suppression of food intake in the ChR2:tdTomatogroup (n=8). FIG. 5b shows the tdTomato control group did not suppressfood intake during photostimulation (n=4) (p=0.06). FIG. 5c showsactivation of LED device (20 Hz light pulses) did not induce a placepreference nor avoidance in both ChR2 and tdTomato groups (n=7 pergroup) (p=0.31). FIG. 5d shows photoactivation of Calca+gastric vagalafferents decreased time spent in center (n=7 per group). FIG. 5e showsmice were exposed to a novel sucrose solution on Day 1 followed byoptogenetic activation of vagal sensory fibers (20 Hz). On Day 5, micewere water-restricted overnight and then given simultaneous access to abottle of sucrose and a bottle of water. The graph is the sucrosepreference score (ChR2, n=7; tdT, n=5). Experimental results are fromone cohort of animals. Bar graphs are mean±SEM. Statistical comparisonswere made using two-way repeated-measures ANOVA, Tukey's post hoc,except for FIG. 5d and FIG. 5e , which were two-tailed t test; **p<0.01,***p<0.001.

DETAILED DESCRIPTION

It is to be understood that the following disclosure provides manydifferent embodiments, or examples, for implementing different featuresof various embodiments. Specific examples of components and arrangementsare described below to simplify the disclosure. These are, of course,merely examples and are not intended to be limiting. The sectionheadings used herein are for organizational purposes and are not to beconstrued as limiting the subject matter described.

The nervous system consists of transcriptomically distinct neuronal celltypes that reflect differences in sensing capabilities, connectivity,and function. Mapping their respective functions represents one of themajor goals and challenges for modern neuroscience. To this end,optogenetics has facilitated the untangling of neural networks by usinglight-activated, genetically encoded opsins to selectively manipulatethe activity of distinct neuronal cell types with spatial precision.However, this functionality has been limited to the brain due toconstraints associated with peripheral light delivery: body tissuestypically lack a stable interface for securing fiber optics and theinflexible nature of these optical probes would cause shearing oftissues and nerves during an animal's natural movements. As a result, acell-type-specific understanding of the peripheral nervous system infreely behaving animals is severely lacking.

This is exemplified by the vagus nerve, which provides the only directneural communication between internal organs and the brain. Peripheralendings of vagal afferent fibers respond to a broad array of stimuli,including hormones, osmolytes, changes in pH, and mechanical distentionthat have diverging functions and contributions to behavior. All of itsdiverse sensory cell bodies reside together within the nodose ganglia,but conventional viral and transgenic methods for targeting geneticallydistinct neuronal populations do not permit organ-specificmanipulations. Although pioneering studies have used fiber optics tooptogenetically manipulate mouse vagal afferents with organ specificity,these studies were conducted under anesthesia to investigate autonomicfunctions. Studying functions beyond reflexes, such as gastrointestinalmechanisms of satiation, requires a more flexible approach. Given thewidespread interest in using vagal nerve stimulation for treatingobesity and other neurological disorders, a key priority for thisresearch field is to attain cell-type- and organ-specific manipulationsof the vagus nerve in animals that are awake. Accordingly, developmentof a biocompatible, wireless optogenetic device for organ-specific lightdelivery was sought after.

Advances in wireless technologies have enabled the internalization oflight sources, bypassing physical constraints associated withfiber-optic cables, and driving a shift in what is possible withoptogenetics. Wireless, radio frequency (RF)-powered devices wereminiaturized for microscale light-emitting diode (μLED) insertion intothe brain and recently encased within stretchable and impermeabletethers for securing them onto subdermal tissues. Despite thesedevelopments, organ-restricted illumination remains a challenge. Awirelessly powered μLED that is secured to the rat bladder using acircumferential elastomer sleeve could enable a similar level offunctionality, but this approach impedes organ expansion. Efforts towirelessly manipulate neural organ function in awake mice includestudies that sutured an μLED onto the heart surface for pace making orintestine surface for controlling colonic motility. However, thesedevices were not described as being functional for >8 days, a limitationfor conducting behavioral studies given the extended recovery periodsrequired after thoracic and abdominal device implantation. Moreover,affixing the μLED to the target organ surface results in lightbackscatter and non-specific optogenetic illumination of nearby tissues.No device has yet enabled chronic and durable cell-type-specificoptogenetic manipulation of peripheral neurons inside of an organ.

Here, the development of a durable, multimodal, wireless platform thatenables optogenetic stimulation of peripheral neurons within organs in ahigh-performance manner is described. The miniaturized wireless deviceis fully implantable, utilizing a soft, thin, and low-modulus tether fortargeting a μLED inside an organ. A unique fabrication method isemployed to make a robust, μLED-housing tether, permitting long-term (>1month), intimate interfacing with peripheral nerve endings in freelybehaving mice. These optogenetic implants can selectively, andindependently, manipulate peripheral nerve activity within multipletarget organs in the same animal using a monolithic design. In addition,a channel isolation strategy is introduced for powering multiple cagesusing a single RF transmitter. Coupled with an advanced coil-antennaapproach, a single telemetry system provides reliable wireless power ineight individual homecages, overcoming cage limitations of otherwireless and fiber-optic-based systems. Precise targeting of a μLEDwithin the stomach revealed an unexpected role for putative, gastricchemosensors in suppressing appetite and revealed a valence mechanism bywhich appetite suppression occurs.

Reference will now be made to more specific embodiments of the presentdisclosure and data that provides support for such embodiments. However,it should be noted that the disclosure below is for illustrativepurposes only and is not intended to limit the scope of the claimedsubject matter in any way.

Organ-Specific, Wireless Gastric Optogenetic Device. FIG. 1 is anillustration of a fully implantable wireless device 100. FIG. 1 showsthe general strategy for targeting a μLED 106 inside a stomach 108. Thedevice 100 includes an analog, front-end electronic circuit 102 for RFharvesting (5.5 mm radius and 1 mm thickness) and a tether 104electrically coupled to circuit 102 that supplies current to μLED 106.Device 100 harvests RF energy from a remotely located wireless RF-powersystem, converts the harvested RF energy into optical energy, andilluminates targeted regions in a stomach 108. μLED 106 is situated inthe middle (between proximal and distal ends of tether 104) rather thanthe distal end of tether 104, allowing tether 104 to be threaded in andout of stomach 108 and secured at two contact points 110 a, 110 b (i.e.,the distal end of tether 104 is threaded into and then out of stomach108 such that μLED 106 is positioned within stomach 108). In someaspects, tether 104 is secured with purse-string sutures. Tether 104 isan ultra-thin tether (0.4 mm wide by 0.2 mm thick), and is more thanthree times smaller than insulin syringe needles used forintraperitoneal injections and tubing used for intragastric infusions.

Features that allow for long-lasting operation of the tether 104 are apre-curved, sandwiched construction. In one aspect, the harvester andμLED were connected with thin copper (12 μm) electrical interconnects ontop of flexible and durable polyimide (18 μm) substrate, and then coatedwith a biocompatible silicone polymer, polydimethylsiloxane (PDMS).However, this design exhibited poor durability and post hoc analysisrevealed μLED tether damage likely caused by a mechanical strain. FIGS.2a-2e illustrates an assembly process for device 100. FIG. 2aillustrates a housing 103 that is configured to house electronic circuit104. To increase durability of device 100, μLED 106 is sandwiched inbetween first and second copper/polyimide bilayers 112 a, 112 b, whichalso provided additional electrical contact (FIGS. 2b and 2c ). Tether104 is coated with silicone while tether 104 is in a curved position(pre-curved) to decrease strain compared to a tether that was coated ina flat orientation and then bent when securing it inside the stomach(post-curved). This was achieved by suspending tether 104 in a bentposition, then applying (e.g., pipetting) small amounts of meltedsilicone around a pre-curved portion 114 that includes μLED 106, andcoating the remaining components using a simple dipping process (FIG. 2d). This resulted in a thin, soft, and lightweight (˜380 mg), wireless,gastric optogenetic implant 100 (FIG. 2e ). The compliant, low-modulusproperties eliminated constraints on the natural motions of the animalwhile also minimizing mechanical strain at the connecting joints.

Three-dimensional (3D) modeling of the mechanics showed that the maximumstrain in the copper traces and PDMS coating of the pre-curved tether(<800 Pa) was dramatically reduced compared to the post-curved tetherstrain (<6600 Pa). Tether 104 was further optimized by mechanicallytesting various curvatures and identified a pre-curved configurationthat was functional beyond 200 kilocycles (kc) (FIG. 3a ). Lifetimemechanical cycle tests with a significant load (0.03 kgF) revealed thatthe pre-curved structure with a radius of 1.15 mm was functional for 200kc, a nearly 10-fold improvement compared to the post-curved structure(FIGS. 3b and 3c ). Although there was improved durability withpre-curved structures that had a radius of 2.87 and 0.72 mm, they werenot as durable as 1.15 mm, likely because 2.87 mm is too similar to theflat structure, whereas the sharp angle with a radius of 0.72 mminterfered with the μLED contact with the pad. Device 100 was alsosubjected to waterproof testing by submerging device 100 into a heatedsaline solution, revealing that it remained continually functional forover 2 months, even in extreme temperatures (e.g., over two months at atemperature of 60° C.).

Heat dissipation is another factor that can limit device functionalitysince nerve endings in the gastrointestinal tract can be temperaturesensitive. Thermal assessment of the device 100 demonstrated minimaltemperature increases (˜0.2° C.) during typical operating conditions (10and 20 Hz with 5 ms light pulse; 10 and 5% duty cycles). Consistent withthis, calculation of specific absorption rate (SAR) using afinite-element method analysis tool showed that the SAR distributionagainst localized RF exposure is below IEEE guidelines. Finally, testsin mice showed that the pre-curved, sandwiched tether was functional forover a month, while the post-curved structure stopped working 3 daysafter implantation (FIG. 3d ).

Efficient and High-Throughput, Wireless Optoelectronic Systems. Thepractical use of optogenetics depends on reliable and cost-effectivelight delivery in multiple animal subjects. A complete laser-based,optogenetics setup remains cost-prohibitive for many labs, given thateach animal subject requires a laser, fiber-optic cannula, fiber-opticpatch cord, and rotary joint to decrease physical constraints of a patchcord. Wireless optogenetics is similarly limited, typically requiring asingle RF-power generator for each homecage. Multiple RF-powergenerators can be used, but they must be operated at least 1 m apartfrom each other to avoid electromagnetic interference. Together, theseconstraints limit the group sizes used for studies, restrict theduration and type of behavioral experiments that can be conducted andoverall prohibit the high-throughput utilization of optogenetics.

To overcome these limitations, a multiplex approach to power eightindividual cages with a single RF-power generator was developed. Thewireless telemetry system has an RF-power generator, controller, RFmultiplexer, decoupling multiplexer, and an antenna set for each of the8 cages; each antenna set is made of a pair of top and bottom coilstructure. Simultaneous and independent control of the eight cages isachieved with coupling and decoupling circuits that manage the tuning ofantennas to operational (13.56 MHz) and non-operational (100 MHz) devicepowering frequencies. For example, when the controller selects antennaset 6, the RF multiplexer tunes antenna set 6 to 13.56 MHz anddecoupling multiplexer detunes the other antenna sets to 100 MHz. Theother antennas that are detuned and deactivated can only pass anegligible amount of energy at a frequency of 100 MHz, whichsignificantly deviates from the resonant frequency of 13.56 MHz.Therefore, the other seven antenna sets do not cause interference evenwhen directly adjacent to the actuating 13.56 MHz antenna; this wasconfirmed with electromagnetic simulation results and validationexperiments in vivo. Since optogenetics typically requires briefintermittent light pulses to avoid depolarization block, this strategycan be used to toggle between multiple cages to deliver intermittentlight pulses. Therefore, the limiting factors for the number of cagesthat can be operated simultaneously with a single RF-power generator arethe stimulation frequency and duration of light pulses. With theproposed arrangement, experiments were conducted within eight cagessimultaneously using 20 Hz and 5 ms pulse duration stimulationparameters. This extends the high-throughput utilizing optogenetics todo the experiments with at least eight mice (e.g., a mouse per cage), atypical group size, at that same time. For example, measurements of foodintake require 4 h for each animal in the group. To complete theanalysis of feeding behavior for two groups of animals (experimental andcontrol), each of which has eight animals, it only takes 8 h, whileapproaches using existing wireless TX system, a single power sourcecoupled with a single cage, demands 64 h (8×8 h). This makes it lessideal for longitudinal experiments, in particular those required formost obesity experimental designs, where a device needs to bechronically implanted for >2 months. In addition, through themodification of the multiplexer board and controller, simultaneousactivation of 16 cages at 10 Hz or 32 cages at 5 Hz frequency ispossible when using a 5 ms light pulse duration.

In addition to couplings, wireless coverage remains a limitation foroptogenetic experiments. Conventional systems utilize a single RFantenna below or around the sides of a homecage. Due to electromagneticdissipation away from the RF source, wireless coverage can be as low as30% in a homecage and worse in larger behavior boxes. Previously, theselimitations were circumvented by increasing RF power, but this resultsin undesired RF energy to animal tissues and increased heat generation.A recent study utilized an RF multiplexer that rapidly toggled powerbetween two antennas to increase coverage. However, this approachrequires careful operation and validation to avoid electromagneticinterference between the antennas, and limits the use of a multiplexerfor powering multiple cages. Here, a simple dual-coil-antenna system forincreasing wireless coverage was introduced. It has a top antenna coilthat is connected to an RF generator and an unconnected antenna coilbelow the cage that passively attracts RF signals towards the animalsubject and cage bottom. Three-dimensional electromagnetic modelingsuggested that the dual-coil-antenna system could enable continuousoperation throughout a location of interest. This was confirmed withlight-power-output measurements of wireless devices at fiverepresentative positions and heights from the cage bottom, whichdemonstrated robust device activation throughout the volume of a cage.Furthermore, the dual-coil-antenna system eliminated the dependence oftransmitted power on the relative orientation angle between thetransmission antenna and the device. Comparison studies furtherindicated that the proposed antenna system outperforms other existingsystems, offering virtually complete wireless coverage in a homecage.

Scalable, Multimodal Device Operation. Multimodal device operation isanother strategy for increasing the efficiency and throughput ofwireless optogenetic studies. Targeting multiple organs with a singledevice could enable multiorgan analysis in the same animal or even beused to examine organ-to-organ interactions. Realization of multimodaltools requires an actuation mechanism that can remotely manage channelselection. Previous efforts utilized higher operating frequencies,microcontroller chip, or Bluetooth® kits for actuating separatechannels, but these approaches require increased RF power (tens of mW)for operation and render them energy-hungry devices. Here, a reed switchwas used in the device that responds to the pattern of externallyapplied electromagnetic RF pulses. In this example, a pulse width longerthan 100 ms triggers the transition from a green μLED to a blue μLEDlocated on a separate tether. The actuation threshold can be adjusted bypairing different capacitors and resistors with the reed switch toprevent unwanted activation or deactivation, and could theoretically betuned for switching between more than two channels. This strategy onlyrequires 10 μW for channel selection, which is 100-fold less power thanother approaches. When combined with the dual-coil antenna and multiplexcoupling/decoupling, the proposed optoelectronic system enables robust,ultra-efficient wireless powering of optogenetic devices in multipleorgans and multiple cages with independent and simultaneous control.Time slots allocated for each cage and threshold pulse for channelselection are tunable, suggesting many scenarios of multiplexing andmultimodal operation. For example, the threshold pulse was set foractivation/deactivation of channels to 100 ms and allocated 250 ms foreach cage. This provides enough time for an implant in each cage toswitch its channel (from Ch1 to Ch2 or vice versa). It requires only 2 s(8×0.25 s) for the switching operation of implants in cages. Next, theTX system can adjust time slots, depending on stimulation conditions.

Optogenetic Manipulation of Gastric Vagal Sensory Endings. To determinethe utility of the optoelectronic system, the role of stomach vagalafferent endings in feeding behavior was investigated. This began byanalyzing the μLED light spread and identifying RF powering parametersneeded for organ specificity. As expected, securing μLED inside thestomach significantly restricts light spread, in contrast to surfaceaffixation, which results in light backscatter intensities well abovethe threshold for opsin activation (FIG. 4a ). Determining whetherimplantation of the stomach device is well tolerated was examined byshowing that ad libitum food intake of mice implanted with the devicewas the same as sham-operated mice (FIG. 4b ). These results indicatethat the wireless device should allow precise optogenetic manipulationsin awake, behaving mice.

A recent study identified genetically distinct vagal afferent neurons inthe nodose ganglion that innervate the stomach and express either Calca,Sst, Gpr65, or Glp1r genes. In contrast to Sst and Gpr65, which exhibiteither mechanosensitive morphological endings in muscle layers (Glp1r)or chemosensitive endings in the mucosal layer (Sst and Gpr65), Calca+neurons form spatially restricted chemosensitive mucosal endings in thecorpus vs. mechanosensitive intramuscular arrays in the stomach antrum.Identification of a role for stomach chemosensation in appetite controlhas been elusive; therefore, the wireless device was used to selectivelyactivate Calca+vagal afferent chemosensitive endings in the corpusregion of the stomach.

To gain cell-type specificity, AAV9 was injected into the nodoseganglion of Calca^(Cre) transgenic mice to introduce Cre-dependentChR2:tdTomato opsin expression or a control group with just tdTomatofluorescent reporter. Precise anatomical specificity was achieved byimplanting the μLED into the fundus, immediately adjacent to the corpus.While Calca+vagal afferents do not innervate the fundus, the device wasimplanted away from the antrum to avoid activation of mechanosensitivefibers. Several weeks after recovering from device implantation, micewere fasted overnight and re-fed the following morning. Compared to nostimulation (RF antenna off), optogenetic activation produced robustsuppression of food intake during refeeding, with greater stimulationfrequencies almost completely suppressing intake (FIG. 5a ). Activationof the device in the control group without ChR2 did not alter feedingbehavior, indicating that RF signals and activation of the device initself do not influence feeding (FIG. 5b ). To further establish thatthe appetite suppression was due to activation of vagal afferent endingsin the stomach, these results were compared to separate cohorts of miceimplanted with six non-attached μLEDs in the abdomen. Althoughoptogenetic stimulation of vagal afferents in this manner suppressedfeeding, the effect was not as robust despite stimulating with six μLEDsrather than a single μLED directly implanted inside the stomach.Furthermore, the non-anchored LED approach required increased operatingpower to compensate for light dissipation, resulting in greater heatgeneration and potential tissue damage. Thus, the gastric optogeneticimplant enables more robust optogenetic activation using less wirelesspower.

Appetite suppression can be associated with positive valence,potentially due to the removal of aversive hunger signals, or aversionin response to harmful stimuli, such as uncomfortable gastric distensionor food poisoning. To investigate the affective mechanisms by whichCalca+gastric vagal afferent neurons might suppress appetite, oversizeddual-coil antennas for robust optogenetic activation in various behaviorboxes were constructed. In one assay, mice were placed in a two-chamberbox with RF power only in one chamber to determine whether mice form anaversion or preference for the optogenetic stimulation chamber.Surprisingly, differences in place preference or avoidance was notobserved (FIG. 5c ), similar to optogenetic stimulation of other vagalafferent cell types that innervate the gastrointestinal tract.Conversely, an open-field assay demonstrated that optogeneticstimulation reduced the time mice spent in the center, indicative ofanxiety-like behavior and suggesting that activation of Calca+gastricvagal afferent fibers might be aversive. The locomotor activity fromopen-field and place-preference tests were analyzed, which revealeddecreased locomotion during optogenetic activation of Calca+gastricvagal afferent; presumably, the mice feel aversion and have decreasedmotivation. In addition, gastrointestinal signals are closely associatedwith taste-sensory signals, suggesting that a learned foodpreference/aversion assay might be most indicative of the mechanismunderlying appetite suppression. To test this hypothesis, mice werehabituated to overnight water restriction for several days and thengiven access to 5% sucrose solution, followed by optogenetic stimulationof Calca+vagal afferent fibers for 4 h. Three days later, mice wereoffered the choice of water or 5% sucrose. This two-bottle preferencetest revealed that activation of stomach Calca+vagal afferentsconditioned mice to avoid the sucrose solution (FIG. 5e ). This suggeststhat appetite suppression occurs via a negative valence mechanism thatalters taste preferences. These results identified a role for stomachmucosal Calca+vagal afferents in appetite suppression and revealed amechanism by which appetite suppression occurs.

While many classical studies have established an important role forvisceral signals in controlling behavior, these surgical- andchemical-denervation experiments lacked organ specificity and did notreveal the identity of sensory neurons that can serve divergingfunctions. Here, wireless μLED devices were developed that permitorgan-specific, optogenetic manipulations, and an ultra-efficientwireless telemetry system for powering multiple cages. The miniaturizedwireless device enabled precise optogenetic stimulation of geneticallydefined vagal afferents innervating the mouse stomach, revealing afunction for Calca+mucosal sensory endings in suppressing food intakevia a negative valence mechanism. The pre-curved sandwich constructionsignificantly extended the lifespan of the μLED device and allowed fortesting various stimulation parameters and behavior tests within thesame subjects. It is envisioned that the current device could be used tooptogenetically manipulate neural circuits throughout thegastrointestinal tract and other hollow organs, such as the bladder withlittle or no modifications.

Prior methods for optogenetic activation of vagal afferents in awakemice have either lacked organ specificity or involved gut injections ofa retrogradely transported opsin virus and fiber-optic implantation inthe hindbrain where vagal afferents terminate. While the latter providesorgan specificity, retrograde viruses can be limited by tropism andincomplete infection of certain cell types. Moreover, vagal afferentsexpress neuropeptides in their peripheral endings which are hypothesizedto be released in the gut to exert efferent functions. In other systems,such as for somatosensation, the peripheral release of neuropeptides byafferent fibers can contribute to behaviors by sensitizing otherafferent subtypes to ongoing stimuli. Finally, fiber optics cannot beused for optogenetically manipulating the enteric nervous system norsplanchnic sensory afferents, which synapse in the spinal cord.Investigating the function of these neural circuits and hypotheses,therefore, requires peripheral optogenetic stimulation that is nowpossible with the proposed wireless gastric optogenetic implant.Multimodal features could further enable the investigation of peripheralinteractions by using different colored μLEDs to activate correspondingcolor-sensitive opsins expressed by separate neural substrates ormultiple organs simultaneously/independently.

In addition to extending optogenetic functionality to the peripheralnervous system, advancements in wireless telemetry were introduced thatgenerally improve the scalability and usability of optogenetics. Thedual-coil-antenna system, which enables reliable and complete wirelesscoverage, is easily constructed using inexpensive copper wire securedonto cardboard or plastic backing. The multiplexing approach furtherallows for the testing of large experimental cohorts, which wasparticularly important for our studies because of the extended durationof feeding behavior tests. This system can be set up in under an hour,is simple to operate, and dramatically decreases the cost and timerequired for conducting optogenetic experiments. Furthermore, thewireless telemetry system has broad applicability for poweringoptogenetic devices in the periphery, brain, or other wireless devices,such as those that measure bioelectrical signals.

Future studies may take advantage of these enabled wirelessoptoelectronic features to chronically activate neural circuits fordays, weeks, or even months. Because adaptations can occur withsustained activation of a neural pathway, such experiments are importantfor investigating the persistence of long-term, physiological effects,including weight loss. This can enable experiments that determinewhether appetite suppression induced by gastric vagal afferentactivation is attenuated in obese mice and whether chronic activation ofvagal afferent endings in the stomach can reverse obesity.Identification of viscerosensory pathways that can either suppress orstimulate appetite will have direct clinical importance for potentiallydeveloping novel therapeutic targets for treating appetite disorders.

Device Fabrication. The process began with flexible copper/polyimide(Cu/PI) bilayer films (thickness; 12 μm/18 μm, AC181200RY, Dupont™Pyralux®) mounted onto a glass slide (dimensions, 5.08 cm×7.62 cm).Then, 2.5-μm thickness of photoresistor was deposited on the Cu/PIsubstrate (AZ 1518, AZ®, recipe; spin coated at 3000 r.p.m. for 30 s),and UV photolithography was used to define patterns for pads andinterconnections (EVG610, EV Group, recipe; UV intensity for 200 mJcm-2). This was followed by immersion in developer solution (AZDeveloper 1:1, AZ®) for 30 s and rinses in distilled water for 10 s.Immersion in copper etchant (LOT: Z03E099, Alfa Aesar™) for 7 min andrinses with acetone, methanol, isopropanol, and distilled water for 1min yielded Cu interconnections and pads on the flexible substrate (FIG.2a ). After samples dry, chip components were mounted, including a μLED,passive components, and IC components using a soldering machine. Anadditional PI/Cu layer (18 μm/12 μm thick) with the bottom chip-mountedCu/PI substrate formed a sandwiched structure (PI/Cu/Cu/PI) (FIGS. 2band 2c ). For encapsulations, a small amount of PDMS (Sylgard™ 184Silicone Elastomer Kit, Dow®; 10:1 mix ratio) was applied using apipette while a clamp held the body of a sample to form a thin,pre-curved, sandwiched structure (FIG. 2d ). Then, the body of a samplewas encapsulated with PDMS by a dip-coating process (500 μm thick).Samples were cured in a vacuum oven at 100° C. for 1 h. These proceduresyield a soft, low-power, wireless gastric optogenetic implant with apre-curved, sandwiched tether (FIG. 2e ).

Finite-Element Method Analysis. For numerical electromagneticsimulations of the proposed device, a finite-element method analysistool (Ansys Elec-tromagnetics Suite 17-HFSS, Ansys®) with Cole-Coledielectric relaxation model where characteristics of biological tissueswere described as a function of frequency was used. Organ systems andtissues of a mouse were modeled to one million meshes for numericalsimulations, and antenna coils made of copper stripes or wires weremodeled to materials with finite conductivity, 58 MS s⁻¹. Forthree-dimension modeling of the mechanics for the devices, a commercialfinite-element method analysis tool (Abaqus/CAE 2018, Dassault Systems)was used to investigate strain effects on the pre-curved and post-curvedstructures. The following parameters were used for simulations:thickness 500/18/12/12/18/500 μm (PDMS/PI/Cu/Cu/PI/PDMS) for thepre-curved structure and 510/12/18/510 μm (PDMS/Cu/PI/PDMS) for thepost-curved structure; elastic properties Young's modulus(MPa)/Poisson's ratio: 1/0.49 for PDMS, 119000/0.34 for Cu, and2500/0.34 for PI. Cu/PI layer was modeled as a composite shell element(S4R). PDMS was modeled as a solid hexahedron element (C3D8R) in thepre-curved structure and as a shell element (S4R) in the post-curvedone.

Mechanical, Optical, and Electrical Measurements. A gauge-force machine(ESM303 Forced Test Stand, MARK-10) was used to perform device lifetimecycling tests with a significant load extended over a period of time(>200 kc) for the pre-curved, post-curved, and pre-curved structureswith three different curvatures (0.72, 1.15, and 2.87 mm) of a tether.The experiments involved the application of strain in three differentdirections: (1) the x-direction, (2) the y-direction, and (3) thez-direction, respectively. After completion of each 1000 cycles, awireless device was immersed in 10% phosphate-buffered saline (PBS)solution for 10 min and measured light intensity using a light meter(LT300, Extech). This test was repeated until a device stoppedfunctioning (FIG. 3b, 3c ). Accelerated life testing was also performedwhere a device was immersed in 10% PBS solution and light intensity wasmonitored as a function of time at various temperatures (25, 60, and 90°C.). For thermal assessments of wireless devices, an infrared camera(VarioCAM HDx head 600, Infra-Tech) was used. Light intensity was fixedat an optical intensity of 10 mW mm⁻², which is enough to activatelight-sensitive proteins, and the camera measured variations intemperature when devices were operated with duty cycles of 20, 40, 60,80, and 100%.

Antenna-Coil Fabrication and Wireless, Power-Control System. A 8-gaugebare Cu wire was used for the bottom antenna coil and Cu stripes (0.635mm thick by 2.54 cm wide) for the top antenna coil. The bottom coil wasplaced under a cage while the top coil was situated 8 cm above the cagebottom. Impedance matching using Network Analyzer (ENA Series E5063A,Keysight) with a discrete capacitor component yielded two antenna coils,each of which resonates at 13.56 MHz (the top coil) and 15 MHz (thebottom coil), respectively; these different frequencies offer broadbandwidth and stable coverage. Wireless power-control systems had anRF-power supply (ID ISC.LRM2500-A, FEIG Electronics), matching board (IDISC.DAT-A, FEIG Electronics), RF multiplexer (ID ISC.ANT.MUX.M8, FEIGElectronics), controller (nRF52832 Development Kit, Nordicsemiconductor), and decoupling multiplexer. The controller wasprogrammed a custom C code based on C code libraries from Nordic(nRF5_SDK_13.0.0_04a0bfd) by Keil uVision 5 IDE (μVision V5.23.0.0).

Measurements of Wireless Coverage. A wireless device was implanted overthe skull, under the skin of a mouse, and their behaviors was recordedusing three cameras (C615, Logitech). A red-colored μLED was embedded inan implanted device to serve as a signal that can be easily detected bycameras over a cage and the wireless TX system transmitted RF signals at1 W. One camera was positioned above a cage and two cameras recordedfrom left and right sides. They recorded behaviors of an animal in acage for 2 min and images were extracted from the recordings and wereanalyzed frame by frame to determine whether an image had capturedwireless operation of a device (red μLED). Next, the number of framesmissing wireless operation was counted. For the purpose of visualdemonstration of wireless coverage, 3D continuous traces of a red μLEDwas reconstructed from extracted images using custom scripts in Python(version 3.7.3-64 bit, Spyder 3.3.6 IDE). The procedures described abovewas repeated for other wireless antenna technologies. For validations ofwireless power TX systems, an electromagnetic probe (TBPS01-TBWA2/40 dB,Tekbox) was used to measure the output power at five representativepositions (A, B, C, D, and E) and various heights from the bottom of theenclosure as a function of the distance and angle.

Mice. Calca^(Cre):GFP homozygous mice (C57Bl/6 background; Jacksonlaboratory Calcatm1.1(cre/EGFP)Rpa) were bred with C57Bl/6 mice togenerate Calca^(Cre): GFP/+heterozygous mice used in experiments.Following surgery, mice were singly housed with ad libitum access tostandard chow diet (LabDiet 5053) in temperature- andhumidity-controlled facilities with 12 h light/dark cycles. Both maleand female mice were used for behavioral experiments. All animal careand experimental procedures were approved by the Institutional AnimalCare and Use Committee at the University of Washington.

Organ-Specific, Wireless, Gastric Optogenetic Device Implantation. Undersurgical anesthesia (isoflurane, 1-2% inhalation), the animal's ventralside was shaved, sterilized with three alternating scrubs of betadineand alcohol, and the surgical field was restricted with sterile drapes.With the animal on its back, a 2 cm skin incision was made along theabdominal midline from the xiphoid cartilage extending to themid-abdomen, and a second cut into the abdominal wall exposed thestomach for device implantation. Ringed forceps were used to gentlygrasp the fore-stomach and pull it out of the abdominal cavity ontogauze soaked with sterile saline. Fine-tipped Dupont forceps were thenused to puncture the stomach fundus and thread the μLED tether in andout of the stomach. With the μLED inside the stomach, the tether wassecured in place with purse-string sutures (5-0 PGA). The deviceharvester was then placed in the abdominal cavity and the stomach wasplaced back into its normal orientation. The abdominal wall was closedwith interrupted stitches using absorbable suture (5-0 PGA), and theskin with non-absorbable suture (6-0 silk). Mice received analgesicsduring the surgery (ketoprofen, 5 mg kg⁻¹) and daily post-operative care(provided with hydrating gel, monitor food intake, and body weight). Formultimodal device implantation, an incision was made in the abdominalcavity and the device was implanted with the blue LED positioned towardsthe thoracic cavity and the green LED towards the abdominal cavity.After recovery from surgery, all animals received daily post-operativecare and monitoring.

Meal-Pattern Analysis. To examine whether mice tolerate stomach deviceimplantation, one group of mice was implanted with the device, whereasanother group underwent a sham surgery where the abdomen was opened nearthe stomach, but it was not punctured nor implanted with a device. Twoweeks after the surgeries, mice were placed in food-monitoring homecages(BioDAQ, v. 2.2). Feeding records were analyzed using BioDAQ Viewer(software v. 2.2.01). A feeding bout (>0.01 g) was defined as a mealif >0.06 g of food was ingested and if it was separated from anothermeal by >5 min.

Nodose Ganglion Injection. With the mouse under anesthesia (isoflurane,1-2% inhalation), a 1-2-cm-long skin incision was made from the leftclavicle going upwards to the animal's jaw. The left vagus nerve wasexposed by separating the platysma, sternohyoideus, and omohyoideusmuscles using blunt dissection. After visualization of the ganglion, thevirus (200 nl) was injected with a glass micro-pipette attached to aNanoject II. The experimental group received AAV9-DIO-ChR2:tdTomato andcontrol groups AAV9-DIO-tdTomato. The skin was then closed withinterrupted stitches (6-0 silk suture). After the experiments, mice wereanesthetized (Beuthanasia, 320 mg kg⁻¹ delivered intraperitoneally) andintracardially perfused with PBS, followed by 4% paraformaldehyde.Brains, nodose ganglion, and stomach tissues were then extracted, postfixed in 4% paraformaldehyde overnight, and cryoprotected in PBScontaining 30% sucrose until the tissues sunk in the sucrose solution.Coronal cryostat sections for the brain, nodose, and stomach tissue werecollected (30, 20, and 10 μm thick), directly mounted onto microscopeslides, and coverslipped using DAPI Fluoromount-G mounting medium(SouthernBiotech). Two mice injected with AAV9-DIO-ChR2:tdTomato wereexcluded from behavioral analysis due to little (2-3 neurons infectedper section) or no virus infection as determined by visualizing thefluorescent reporter, tdTomato.

In Situ Hybridization. Single-molecule fluorescence in situhybridization was performed using an RNAscope Fluorescent Multiplex Kit(Advanced Cell Diag-nostics). C1 and C3 DNA oligonucleotide probes weredesigned for Calca and tdTomato. Nodose sections were fixed in 4%paraformaldehyde for 15 min and then washed in 50, 70, 100, and 100%ethanol for 5 min each. Slides were dried for 5 min. Proteins weredigested using protease solution (pretreatment solution 3) for 60-90 s.Immediately afterward, slides were washed twice in PBS. In parallel, C1and C2 probes were heated in a 40° C. water bath for 10 min. Probes wereapplied to the slides, which were coverslipped and placed in a 40° C.humidified incubator for 3 h. Slides were rinsed twice in RNAscope washbuffer and then underwent the colorimetric reaction steps according tothe standard kit protocol. After the final wash buffer, slides wereimmediately coverslipped using DAPI (4′,6-diamidino-2-phenylindole)Fluoromount-G mounting medium. Images were captured using alaser-scanning confocal (FV1200, Olympus) and epifluorescent (EclipseE600, Nikon) microscopes.

Measurement of μLED Light Spread. Light intensity measurements (PM100D,Thorlabs) were acquired from fresh stomach tissue preparations ex vivo.Measurements were recorded after implanting the LED inside the stomachor after suturing the tether to the stomach surface with the LEDdirected towards the organ; the light sensor (S130C, Thorlabs) wasencased in saran wrap and placed directly over the LED tether housing.2, 4, 6, 8, 10, and 12 W antenna power outputs were tested. In aseparate experiment, with LED not attached to a stomach, lightdissipation was measured by placing the sensor 0, 0.5, and 1 cm awayfrom the front or backside of the LED housing.

Fasting and Refeeding Experiments. Mice were food restricted overnight(16 h) and refed the following morning. Food intake was manuallymeasured 1, 2, and 3 h after refeeding. The same animals underwentmultiple fasting—refeeding tests to examine different optogeneticstimulation parameters: no stimulation (RF antenna off), 10 Hz, and 20Hz optogenetic stimulation (5 ms pulse width; RF power 4 W). Experimentswere conducted 5 days apart.

Real-Time, Place-Preference (RTTP) Assay. Mice were placed in an RTPPbox consisting of two chambers (20 cm×18 cm) and a small transitionarea. Antennas were installed in both chambers, but only one chamber wasconnected to an RF generator to continuously deliver RF power (20 Hz, 5ms pulse width, 4 W). The time spent in each chamber (20 min trial) wasanalyzed using video-tracking software (EthoVision XT 10, Noldus).

Open-Field Test. Mice were placed in the center of a 40 cm×40 cm squareopen-field arena with non-transparent white Plexiglas. The totaldistance moved and time in the center (20 cm×20 cm imaginary square),during the 10 min trial, were analyzed with video-tracking software withEthoVision. An RF antenna provided wireless power (20 Hz, 5 ms pulsewidth, 4 W) throughout the entire behavior box.

Two-Bottle Flavor Preference Test. Mice were accustomed to drinkingwater from two test tubes that replaced their normal water bottles.After acclimation, mice were water-deprived overnight, and the followingmorning received 30 min access to a novel 5% sucrose solution;immediately following sucrose consumption, mice received 4 h ofoptogenetic stimulation (20 Hz, 5 ms pulse width, 4 W). Three dayslater, mice were water-deprived overnight, and the following morningreceived 30 min access to separate test tubes containing either water or5% sucrose solution. The intake of both solutions was measured andpresented as a preference ratio (5% sucrose intake/total intake ofsucrose and water solutions).

Statistics. Data were analyzed using Prism 5.0 (GraphPad software).Sample sizes were estimated based on prior experience and expectedvariability in feeding behavior. An animal from data analysis wasexcluded if post hoc histological analysis showed no viral transductionas indicated by an absence of tdTomato fluorescence. For graphscomparing two experimental conditions, unpaired two-tailed Student's ttest was used. The data sets (multiple treatments and time-points) wereanalyzed with repeated-measures two-way analysis of variance tests (timerepeated factor) and Tukey's post hoc tests. All data sets wereconducted using Shapiro-Wilk normality test, and all passed thenormality tests.

Although various embodiments of the present disclosure have beenillustrated in the accompanying Drawings and described in the foregoingDetailed Description, it will be understood that the present disclosureis not limited to the embodiments disclosed herein, but is capable ofnumerous rearrangements, modifications, and substitutions withoutdeparting from the spirit of the disclosure as set forth herein.

The term “substantially” is defined as largely but not necessarilywholly what is specified, as understood by a person of ordinary skill inthe art. In any disclosed embodiment, the terms “substantially”,“approximately”, “generally”, and “about” may be substituted with“within [a percentage] of” what is specified, where the percentageincludes 0.1, 1, 5, and 10 percent.

The foregoing outlines features of several embodiments so that thoseskilled in the art may better understand the aspects of the disclosure.Those skilled in the art should appreciate that they may readily use thedisclosure as a basis for designing or modifying other processes andstructures for carrying out the same purposes and/or achieving the sameadvantages of the embodiments introduced herein. Those skilled in theart should also realize that such equivalent constructions do not departfrom the spirit and scope of the disclosure, and that they may makevarious changes, substitutions, and alterations herein without departingfrom the spirit and scope of the disclosure. The scope of the inventionshould be determined only by the language of the claims that follow. Theterm “comprising” within the claims is intended to mean “including atleast” such that the recited listing of elements in a claim are an opengroup. The terms “a”, “an”, and other singular terms are intended toinclude the plural forms thereof unless specifically excluded.

What is claimed is:
 1. An organ-specific wireless optogenetic device,the device comprising: an electronic circuit, wherein the electroniccircuit is configured to harvest and convert radio frequency (RF) energyinto optical energy; and a tether coupled to the electronic circuit andcomprising a μLED, wherein when the device is implanted in a subject theμLED illuminates targeted regions in the organ.
 2. The device of claim1, wherein the electronic circuit is configured to receive the harvestedRF energy from a remotely located RF-power system.
 3. The device ofclaim 2, wherein the RF-power system comprises a dual-coil-antennasystem.
 4. The device of claim 1, wherein the μLED is positioned betweenproximal and distal ends of the tether.
 5. The device of claim 1,wherein a portion of the tether that comprises the μLED has apre-curved, sandwiched configuration.
 6. The device of claim 4, whereinthe pre-curved, sandwich configuration comprises flexiblecopper/polyimide (Cu/PI) bilayer films.
 7. The device of claim 4,wherein the pre-curved, sandwiched configuration comprises PI/Cu/Cu/PI.8. The device of claim 1, wherein a portion of the tether isencapsulated by a composition.
 9. The device of claim 8, wherein theportion of the tether comprises the μLED.
 10. The device of claim 8,wherein the composition comprises silicone.
 11. A method of treatingobesity, the method comprising: implanting an organ-specific wirelessoptogenetic device into a subject, wherein the organ-specific wirelessoptogenetic device comprises: an electronic circuit configured toharvest and convert radio frequency (RF) energy into optical energy; anda tether coupled to the electronic circuit and comprising a μLEDpositioned between a proximal end and a distal end of the tether;activating an RF-power system to produce RF energy; harvesting, by theorgan-specific wireless optogenetic device, the RF energy; converting,by the organ-specific wireless optogenetic device, the RF energy intooptical energy; and illuminating, by the μLED, targeted regions in thestomach of the subject.
 12. The method of claim 11, wherein the targetedregions in the stomach comprise vagus nerve endings.
 13. The method ofclaim 11, wherein the RF-power system is remotely-located from theorgan-specific wireless optogenetic device and comprises adual-coil-antenna system.
 14. The method of claim 11, wherein theorgan-specific wireless optogenetic device is configured with apre-curved, sandwiched configuration.
 15. The method of claim 14,wherein the sandwich configuration comprises flexible copper/polyimide(Cu/PI) bilayer films.
 16. The method of claim 14, wherein thesandwiched configuration comprises PI/Cu/Cu/PI.
 17. The method of claim11, where a portion of the tether comprising the μLED is encapsulated bya composition.
 18. The method of claim 17, wherein the compositioncomprises silicone.
 19. The method of claim 17, wherein the compositionis coated on the portion of the tether while the tether is in a bentposition.
 20. The method of claim 11, where appetite suppression occursin the subject via a negative valence mechanism that alters tastepreferences.